Novel nucleic acid probe and novel method of assaying nucleic acid using the same

ABSTRACT

A novel nucleic acid probe for nucleic acid determination includes a single-stranded nucleic acid labeled with plural fluorescent dyes containing at least one pair of fluorescent dyes to induce FRET, the pair of fluorescent dyes including a fluorescent dye (a donor dye) capable of serving as a donor dye and a fluorescent dye (an acceptor dye) capable of serving as an acceptor dye, in which the nucleic acid probe has such a base sequence and is labeled with the fluorescent dyes so that the fluorescence intensity of the acceptor dye decreases upon hybridization with a target nucleic acid. A novel nucleic acid determination method uses the probe. The probe and method can determine one or more types of target nucleic acids in an assay system in parallel using a simple apparatus.

TECHNICAL FIELD

The present invention relates to, for example, a novel nucleic acidprobe for determining one or more types of target nucleic acids (i.e.,one or plural target nucleic acids) in an assay system and fordetermining such target nucleic acids, if plural, in an assay system inparallel using a simple apparatus and to a method for nucleic aciddetermination using the probe.

More specifically, it relates to a novel nucleic acid probe for nucleicacid determination, which comprises a single-stranded nucleic acid andis labeled with plural fluorescent dyes, which fluorescent dyes includesat least one pair of fluorescent dyes to induce fluorescence resonanceenergy transfer (hereinafter briefly referred to as “FRET”), which pairof fluorescent dyes includes a fluorescent dye capable of serving as adonor dye (hereinafter referred to as “donor dye”) and a fluorescent dyecapable of serving as an acceptor dye (hereinafter referred to as“acceptor dye”). It also relates to a method for nucleic aciddetermination using the nucleic acid probe, to a reagent kit for nucleicacid determination for use in the method, to a method for analyzing ordetermining polymorphism and/or mutation of target nucleic acids, and toan assay kit for use in the method just mentioned above.

BACKGROUND ART

Certain nucleic acid probes comprising a single-stranded oligonucleotideand a pair of fluorescent dyes to induce FRET including a donor dyefluorescein and an acceptor dye X-rhodamine are known in the art [JikkenIgaku (Experimental Medicine), 15, e7, 728-733 (1997)]. These nucleicacid probes are used in monitoring of polymerase chain reaction (PCR)and are not intended to direct determination of nucleic acids.

Some nucleic acid probes have been used for detecting a target nucleicacid (Biochemistry, 34, 285-292 (1995). In this case, the nucleic acidprobe emits fluorescence at a low intensity, as a result of FRET, beforehybridization with the target nucleic acid. Specifically, thefluorescein is prevented from emitting fluorescence and can emitfluorescence only at a low intensity. In contrast, the X-rhodamine isnot so prevented from emitting fluorescence but emits fluorescence at anintensity lower than that of the prevented fluorescence emission of thefluorescein. Upon hybridization with the target nucleic acid, the probechanges in its conformation such that FRET disappears. As a result, thefluorescein emits fluorescence at an increasing intensity to therebyincrease the fluorescence intensity of the whole reaction system.However, if a nucleic acid probe does not change in its conformationafter hybridization, the fluorescence intensity does not change, andsuch a nucleic acid probe is not suitable for use in nucleic aciddetermination.

The present inventors have developed a nucleic acid probe, namely, “afluorescence quenching nucleic acid probe” that decreases itsfluorescence emission after hybridization with a target nucleic acidwithout the aid of another nucleic acid probe (Nucleic acid, 29, No.6e34 (2001); EP 1 046 717 A9; Japanese Patent Publication No.2001-286300A). The nucleic acid probe comprises a single-strandedoligonucleotide labeled with a fluorescent dye in an end region. Thefluorescent dye and the base sequence of the oligonucleotide aredesigned such that the fluorescence intensity decreases uponhybridization with the target nucleic acid. Use of the nucleic acidprobe makes it possible to determine precisely, easily and in short timethe target nucleic acid in a trace amount. However, this conventionalmethod utilizes fluorescence quenching due to interaction betweenbase-pair complex of G (guanine) and C (cytosine) combined through ahydrogen bond and the fluorescent dye upon the formation of adouble-stranded oligonucleotide. In other words, the method utilizesfluorescence quenching due to emission energy transfer from thefluorescent dye to the complex. However, relatively a few type offluorescent dye is subjected to quenching action according to thismechanism, and the method is limited in fluorescent colors that areusable.

When an assay system contains plural types of target nucleic acids,fluorescence colors of the number of color type(s) equal to that of thetypes of the target nucleic acids must be used to determine the targetnucleic acids in parallel. In other words, nucleic acid probes emittingfluorescence of different colors in the number of type(s) equal to thatof the types of the target nucleic acids are needed. In enlargement ofthe number of fluorescent color type(s) in the conventional method, thenumber of type(s) of fluorescent dyes are needed to be larger, whichfluorescent dyes are capable of inducing interaction with the G-Chydrogen bonding pair and are different in emitting fluorescence colors.However, the types of fluorescent colors applicable to the conventionalmethod are limited and thereby the method is limited in determiningplural (specifically three or more) types of target nucleic acids inparallel. In addition, that types of fluorescent dyes are different inthe conventional method means that exciting wavelengths different intypes also are needed, thus requiring different types of excitationlight sources to excite all the fluorescent dyes. The conventionalmethod therefore requires an apparatus of a large-scale and is noteconomical.

Under these circumstances, it is an object of the present invention toprovide a novel nucleic acid probe for nucleic acid determination(“novel nucleic acid probe for nucleic acid determination” may behereinafter briefly referred to as “nucleic acid probe”), which includesa single-stranded oligonucleotide labeled with a pair of fluorescentdyes capable of inducing FRET and including a donor dye and an acceptordye and can precisely and easily determine one or more types (i.e., oneor plural types) of target nucleic acids in small amounts in parallel ina short time using a simple analyzer (instrument). Another object of thepresent invention is to provide a novel method for nucleic aciddetermination using the nucleic acid probe (hereinafter briefly referredto as “nucleic acid determination method”), a reagent kit for use in themethod, a method for determining polymorphism and/or mutation of targetnucleic acids, and an assay kit for use in the method just mentionedabove.

DISCLOSURE OF INVENTION

Under consideration about the fluorescence quenching probe as a basicconcept, the present inventors have made intensive investigations ondifferent nucleic acid probes each including a single-strandedoligonucleotide labeled with different fluorescent dyes at twopositions. As a result, they have obtained findings that, when theoligonucleotide is labeled at specific positions with a specific pair ofa donor dye and an acceptor dye, the fluorescence intensity of theacceptor dye or the fluorescence intensities of both the donor dye andacceptor dye significantly decrease in a probe-nucleic acid complex(including the nucleic acid probe hybridized with the target nucleicacid) after hybridization with the target nucleic acid, which complex isformed by hybridization of the nucleic acid probe with the targetnucleic acid. The present invention has been accomplished based on thesefindings.

Therefor, the present invention provides the following novel nucleicacid probes, methods, kits and devices:

-   1. A novel nucleic acid probe for determining one or more types of    target nucleic acids in an assay system, comprising a    single-stranded nucleic acid being labeled with plural fluorescent    dyes, the fluorescent dyes comprising at least one pair of    fluorescent dyes to induce fluorescence resonance energy transfer    (FRET), the pair of fluorescent dyes comprising a fluorescent dye (a    donor dye) capable of serving as a donor dye and a fluorescent dye    (an acceptor dye) capable of serving as an acceptor dye, wherein the    nucleic acid probe has a base sequence and is labeled with the    fluorescent dyes such that the fluorescence intensity of the    acceptor dye decreases upon hybridization with a target nucleic    acid.-   2. The novel nucleic acid probe as described above under 1 for    determining one or more target nucleic acids in an assay system,    wherein the fluorescence intensities of the donor dye and acceptor    dye decrease upon hybridization with the target nucleic acid.-   3. The novel nucleic acid probe as described above under any one of    1 and 2 for determining one or more types of target nucleic acids in    an assay system, wherein the fluorescent dye capable of serving as a    donor dye is selected from BODIPY FL, BODIPY 493/503, 5-FAM,    Tetramethylrhodamine, and 6-TAMRA.-   4. The novel nucleic acid probe as described above under any one of    1 and 3 for determining one or more types of target nucleic acids in    an assay system, which comprises one pair of the donor dye and the    acceptor dye.-   5. The novel nucleic acid probe as described above under any one of    1 and 4 for determining one or more types of target nucleic acids in    an assay system, wherein the nucleic acid probe is labeled with the    donor dye in an end region and has a base sequence designed such    that, when the probe hybridizes with the target nucleic acid at the    end region, the target nucleic acid has at least one G (guanine) in    its base sequence as a first to third base from its terminal base    hybridized with the probe.-   6. The novel nucleic acid probe as described above under any one of    1 and 5 for determining one or more types of target nucleic acids in    an assay system, wherein the nucleic acid probe has a base sequence    designed such that that plural base pairs of a probe-nucleic acid    hybrid in a region labeled with the donor dye constitute at least    one pair of G (guanine) and C (cytosine) upon the hybridization with    the target nucleic acid.-   7. The novel nucleic acid probe as described above under any one of    1 and 6 for determining one or more types of target nucleic acids in    an assay system, wherein the nucleic acid probe is labeled with the    donor dye in a 5′ end region inclusive of the 5′ end.-   8. The novel nucleic acid probe as described above under any one of    1 and 7 for determining one or more types of target nucleic acids in    an assay system, wherein the nucleic acid probe is labeled with the    donor dye in a 3′ end region inclusive of the 3′ end.-   9. The novel nucleic acid probe as described above under 7 for    determining one or more types of target nucleic acids in an assay    system, wherein the nucleic acid probe has a G or C base at the 5′    end and is labeled with the donor dye at the 5′ end.-   10. The novel nucleic acid probe as described above under 8 for    determining one or more types of target nucleic acids in an assay    system, wherein the nucleic acid probe has a G or C base at the 3′    end and is labeled with the donor dye at the 3′ end.-   11. A novel method for nucleic acid determination, the method    comprising the steps of adding one or more types of the nucleic acid    probes according to any one of claims 1 to 10 into an assay system    containing one or more types of target nucleic acids, the nucleic    acid probe(s) being capable of hybridizing with the target nucleic    acid(s), being in the number of type(s) equal to or larger than that    of the target nucleic acid(s), and emitting fluorescence in    different colors; allowing the nucleic acid probe(s) to hybridize to    the target nucleic acid(s); and determining differential decrease(s)    in fluorescence intensity between before and after hybridization at    wavelength(s) in the number of type(s) equal to or larger than that    of the types of the nucleic acid probe(s).-   12. A kit for determining one or more types of target nucleic acids    in an assay system, wherein said kit includes or accompanied by a    nucleic acid probe as described above under any one of 1 to 10.-   13. A method for analyzing or determining polymorphism and/or    mutation of one or more types of target nucleic acids in an assay    system, which comprises the steps of adding one or more types of the    nucleic acid probes as described above under any one of 1 to 10 into    an assay system containing one or more types of target nucleic    acids, the nucleic acid probe(s) being capable of hybridizing with    the target nucleic acid(s), being in the number of type(s) equal to    or larger than that of the target nucleic acid(s), and emitting    fluorescence in different colors; allowing the nucleic acid probe(s)    to hybridize to the target nucleic acid(s); and determining    differential decrease(s) in fluorescence intensity between before    and after hybridization at wavelength(s) in the number of type(s)    equal to or larger than that of the types of the nucleic acid    probe(s).-   14. A kit for analyzing or determining polymorphism and/or mutation    of one or more types of target nucleic acids in an assay system,    wherein said kit includes or accompanied by a nucleic acid probe as    described above under any one of 1 to 10.-   15. A method as described above under one of 11 and 13, wherein said    target nucleic acids are one or more types of nucleic acids from    cells of a microorganism obtained by single colony isolation or from    cells of an animal.-   16. A method as described above under one of 11 and 13, wherein said    target nucleic acids are one or more types of nucleic acids    contained in cells or a homogenate of cells, wherein cells are ones    from a co-cultivation system of microorganisms or symbiotic    cultivation system of microorganisms.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in further detail with referenceto several preferred embodiments.

Specifically, the present invention provides, as a first invention, anucleic acid probe for determining one or plural target nucleic acids,comprising a single-stranded olygonucleotide being labeled with pluralfluorescent dyes, the plural fluorescent dyes containing at least onepair of fluorescent dyes to induce fluorescence resonance energytransfer (FRET), the pair of fluorescent dyes comprising a donor dye andan acceptor dye, in which the nucleic acid probe has a base sequence andis labeled with the fluorescent dyes such that the fluorescenceintensity of the acceptor dye decreases in a probe-nucleic acid hybridcomplex formed upon hybridization of the probe with a target nucleicacid. Among such nucleic acid probes, preferred probes are designed suchthat the fluorescence intensities of both the donor dye and acceptor dyedecrease upon hybridization of the nucleic acids with the target nucleicacid.

The term “probe-nucleic acid hybrid complex” as used herein means one(complex) in which at least a nucleic acid probe according to thepresent invention, which is labeled with plural fluorescent dyes, and atarget nucleic acid are hybridized with each other. For the same ofbrevity, it will hereinafter be called a “nucleic acid hybrid complex”in a shorten form.

The terms as used herein—such as nucleic acid probes, to hybridize,hybridization, stem-loop structures, quenching, quenching effects, DNAs,RNAs, cDNAs, mRNAs, rRNAs, XTPs, dXTPs, NTPs, dNTPs, nucleic acidprobes, helper nucleic acid probes (or nucleic acid helper probes, orsimply helper probes), to hybridize, hybridization, intercalators,primers, annealing, extending reactions, thermal denaturing reactions,nucleic acid melting curves, PCR, RT-PCR, RNA-primed PCR, stretch PCR,reverse PCR, PCR using Alu sequence(s), multiple PCR, PCR using mixedprimers, PCR using PNA, hybridization assays, FISH methods (fluorescentin situ hybridization assays), PCR methods (polymerase chain assays),LCR methods (ligase chain reactions), SD methods (strand displacementassays), competitive hybridization, DNA chips, nucleic acid detecting(gene-detecting) devices, SNP (single nucleotide polymorphism), andco-cultivation systems of plural microorganisms—have the same meaningsas the corresponding terms generally employed these days in molecularbiology, genetic engineering, bioengineering and the like.

Further, the term “nucleic acid determination” as used herein meansquantification, quantitative determination, qualitative detection, ormere detection of a target nucleic acid.

The term “target nucleic acid” as used herein means and includes anynucleic acid or gene the quantification, quantitative determination,qualitative detection, or mere detection of which is intended,irrespective whether it is in a purified form or not and furtherirrespective of its concentration. Nucleic acids other than the targetnucleic acid may also coexist with the target nucleic acid. An assaysystem contains one or more types of target nucleic acids. The targetnucleic acid may be, for example, at least one specific nucleic acid tobe determined in a co-cultivation system of microorganisms (a mixedsystem containing RNAs or genetic DNAs of plural microorganisms) or in asymbiotic cultivation system of microorganisms (a mixed systemcontaining RNAs or genetic DNAs of plural animals or plants and/or ofplural microorganisms). The target nucleic acid can be purified, ifneeded, according to a conventionally known method. For example, it canbe purified using a commercially available purification kit. Examples ofthe nucleic acids include DNAs, RNAs, PNAs, oligodeoxyribonucleotides,oligoribonucleotides, and chemically modified products of these nucleicacids. Such chemically modified nucleic acids include, for example,2′-o-methyl(Me)-RNAs.

Donor dyes capable of serving as a donor dye in FRET for use in thepresent invention are specified as dyes satisfying at least thefollowing conditions: (a) they are excited at a specific wavelength andemit light at a specific wavelength; (b) they can transfer theirlight-emitting energy to a specific dye (a dye capable of serving as anacceptor dye); and (c) when a G-C base pair complex (a G-C base paircomplex bonded through hydrogen bonds; hereinafter briefly referred toas “G-C hydrogen bond complex” for sake of brevity in some parts of thedescription) is in the vicinity of the donor dye, they can transfer theenergy to the base pair. Any dyes that satisfy these conditions can beused in the present invention. Among them, preferred dyes are thosecapable of serving as a donor dye in FRET, and when a nucleic acid probelabeled with the dye alone hybridizes with the target nucleic acid, theprobe-nucleic acid hybrid complex emits fluorescence at a decreasedintensity (Nucleic Acid, Vol. 29, Nol. 6, e34 (2001))

Illustrative of such donor dyes are BODIPY FL (trade names; products ofMolecular Probes Inc., USA), BODIPY 493/503 (trade names; products ofMolecular Probes Inc., USA),5-FAM, Tetramethylrhodamine, 6-TAMRA,Fluorescein and derivatives thereof [for example, fluroresceinisothiocyanate (FITC) and its derivatives]; Alexa 488, Alexa 532, cy3,cy5, EDANS (5-(2′-aminoethyl)amino-1-naphtalene sulfonic acid),rhodamine 6G (R6G) and its derivatives [for example,tetramethylrhodamine isothiocyanate (TMRITC)], BODIPY FL/C3 (tradenames; products of Molecular Probes Inc., USA), BODIPY FL/C6 (tradenames; products of Molecular Probes Inc., USA), BODIPY 5-FAM(tradenames; products of Molecular Probes Inc., USA),BODIPY TMR (trade names;products of Molecular Probes Inc., USA) or its derivatives [for example,BODIPY TR (trade names; products of Molecular Probes Inc., USA)], BODIPYR6G (trade names; products of Molecular Probes Inc., USA), BODIPY 564(trade names; products of Molecular Probes Inc., USA),BODIPY 581/591(trade names; products of Molecular Probes Inc., USA).

Among them, preferred donor dyes are BODIPY FL (trade name; a product ofMolecular Probes Inc., USA), the BODIPY FL-series dyes (trade names;products of Molecular Probes Inc., USA), BODIPY 493/503 (trade name; aproduct of Molecular Probes Inc., USA), 5-FAM, BODIPY 5-FAM (trade name;a product of Molecular Probes Inc., USA), Tetramethylrhodamine, and6-TAMRA, of which the BODIPY FL series dyes (trade name; a product ofMolecular Probes Inc., USA), BODIPY 493/503 (trade name; a product ofMolecular Probes Inc., USA), 5-FAM, Tetramethylrhodamine, and 6-TAMRA.The donor dyes for use in the present invention are, however, notlimited to these examples.

Acceptor dyes for use in the present invention can be any dyes that arecapable of serving as an acceptor dye in a pair with a donor dye, i.e.,that are capable of receiving energy transferred from the donor dye. Inother words, such acceptor dyes have quenching capability to the donordye. The type of the acceptor dye depends on the type of the donor dyeto constitute the pair. For example, X-rhodamine and BODIPY 581/591(trade name; a product of Molecular Probes Inc., USA) can be used as theacceptor dye when the donor dye is any of the BODIPY FL series dyes(trade name; a product of Molecular Probes Inc., USA), BODIPY FL-seriesdyes (trade names; products of Molecular Probes Inc., USA), BODIPY493/503 (trade name; a product of Molecular Probes Inc., USA), 5-FAM,BODIPY 5-FAM (trade name; a product of Molecular Probes Inc., USA),Tetramethylrhodamine, and 6-TAMRA. However, acceptor dyes for use in thepresent invention are not limited to these examples.

The nucleic acid probe according to the present invention, which is tobe hybridized to the target nucleic acid, may be formed of either anoligodeoxyribonucleotide or an oligoribonucleotide. The nucleic acidprobe may be a chimeric oligonucleotide which contains both of them.These oligonucleotides may be in chemically-modified forms. Suchchemically-modified oligonucleotides may be inserted in chimericoligonucleotides.

Examples of the modified positions of the chemically-modifiedoligonucleotide can include an end hydroxyl group or end phosphate groupof an end portion of an oligonucleotide, the position of a phosphateportion of an internucleoside, the 5-carbon of a pyrimidine ring, andthe position of a saccharide (ribose or deoxyribose) in a nucleoside.Preferred examples are the positions of ribose or deoxyribose. Specificexamples can include 2′-O-alkyloligoribonucleotides (“2′-O-” willhereinafter be abbreviated as “2-O-”),2-O-alkyleneoligo-ribonucleotides, and 2-O-benzyloligoribonucleotides.The oligonucleotide is modified at the OH group(s) on the 2′carbon(s) ofone or more ribose molecules at desired positions thereof with alkylgroup(s), alkylene group(s) or benzyl group(s) (via ether bond(s)).Preferred examples useful in the present invention can include, among2-O-alkyloligo-ribonucleotides, 2-O-methyloligoribonucleotide,2-O-ethyl-oligoribonucleotide and 2-O-butyloligoribonucleotide; among2-O-alkyleneoligoribonucleotides, 2-O-ethyleneoligo-ribonucleotide; and2-O-benzyloligoribonucleotide. Particularly preferably,2-O-methyloligoribonucleotide (hereinafter simply abbreviated as“2-O-Me-oligo-ribonucleotide”) can be used. Application of such chemicalmodification to an oligonucleotide enhances its affinity with a targetnucleic acid so that the efficiency of hybridization with a nucleic acidprobe according to the present invention is improved. The improvedefficiency of hybridization leads to a further improvement in the rateof a decrease in the intensity of fluorescence from the fluorescent dyeof the nucleic acid probe according to the present invention. As aconsequence, the accuracy of determination of the concentration of thetarget nucleic acid is improved further.

Incidentally, it is to be noted that the term “oligonucleotide” as usedherein means an oligodeoxy-ribonucleotide or an oligoribonucleotide orboth of them and hence, is a generic term for them.2-O-alkyloligoribonucleotides, 2-O-alkyleneoligo-ribonucleotides and2-O-benzyloligoribonucleotide can be synthesized by a known process[Nucleic Acids Research, 26, 2224-2229 (1998)]. As custom DNA synthesisservices are available from GENSET SA, France, they can be readilyobtained. The present inventors have completed the present invention byconducting experiments with the compounds furnished by this companypursuant to our order.

Incidentally, use of a nucleic acid probe according to the presentinvention with modified DNA, such as 2-O-methyloligoribonucleotide(hereinafter simply called “2-O-Me-oligoribonucleotide), inserted in anoligodeoxy-ribonucleotide primarily for the determination of RNA,especially for the determination of rRNA can provide preferred results.

Upon determination of RNA by the nucleic acid probe according to thepresent invention, it is preferred to subject an RNA solution as asample to heat treatment at 80 to 100° C., preferably 90 to 100° C.,most preferably 93 to 97° C. for 1 to 15 minutes, preferably 2 to 10minutes, most preferably 3 to 7 minutes before hybridization with theprobe such that the higher-order structure of RNA can be degraded, asthis heat treatment makes it possible to improve the efficiency ofhybridization.

It is also preferred to add a helper probe to a hybridization reactionmixture for raising the efficiency of hybridization of the nucleic acidprobe of this invention to the hybridization sequence region. In thiscase, the oligonucleotide of the helper probe can be anoligodeoxy-ribonucleotide, an oligoribonucleotide or an oligonucleotidesubjected to similar chemical modification as described above. Examplesof the above-described oligonucleotides can include those having thebase sequence of (5′)TCCTTTGAGT TCCCGGCCGG A(3′) as the forward type andthose having the base sequence of (5′)CCCTGGTCGT AAGGGCCATG ATGACTTGACGT(3′) as the backward type or the reverse type. Preferred examples ofthe chemically-modified oligonucleotide can include2-O-alkyl-oligoribonucleotides, notably 2-O-Me-oligoribonucleotide.

Where the base strand of the nucleic acid probe according to the presentinvention is formed of 35 or fewer bases, use of a helper probe isparticularly preferred. When a nucleic acid probe according to thepresent invention longer than a 35-base strand is used, however, it mayonly be necessary to thermally denature target RNA in some instances.

When the nucleic acid probe according to the present invention ishybridized to RNA as described above, the efficiency of hybridization isenhanced. The fluorescence intensity, therefore, decreases correspondingto the concentration of RNA in the reaction mixture so that RNA can bedetermined up to a final RNA concentration of about 150 pM.

The present invention also relates to an assay kit for determining theconcentration of one or more (one or plural) of types of target nucleicacids in an assay system, which includes or carries one or more of thenucleic acid probes of the invention in the number of type(s) equal toor larger than the number of the target nucleic acid(s) and furtherincludes or carries the helper probe.

In RNA determination according to a conventional hybridization methodusing a nucleic acid probe, an oligodeoxyribonucleotide or anoligoribonucleotide has been used as the nucleic acid probe. Because RNAitself has a higher-order firm structure, the efficiency ofhybridization between the probe and the target RNA was poor, resultingquantification of low accuracy. The conventional methods therefore areaccompanied by irksomeness that a hybridization reaction is conductedafter denaturing RNA and immobilizing the denatured RNA on a membrane.In contrast, the method of the present invention uses a nucleic acidprobe a ribose portion of which has been modified to have high affinityto a particular structural part of RNA, so that a hybridization reactioncan be conducted a higher temperature as compared with the conventionalmethods. The adverse effect of the higher order structure of RNA can beavoided only by heat denaturation of the RNA as a pretreatment and thecombination use of the helper probe. By this configuration, the methodof the present invention can yield a hybridization efficiency ofsubstantially 100% and can quantitatively determine the target RNAsufficiently. Further, the method according to the present invention isfar significantly simplified as compared with the conventional method.

The probe of the present invention comprises 5 to 60 bases, preferably10 to 35 bases, and typically preferably 15 to 20 bases. If the numberof base(s) exceeds 60, the permeability of the nucleic acid probethrough cell membranes of microorganisms may become lower when used inan FISH method, thereby narrowing the applicable ranges of the presentinvention. The number of base(s) smaller than 5 tends to inducenon-specific hybridization, thus resulting in a large determinationerror.

The nucleic acid probe is labeled with the donor dye in an end region,and the base sequence thereof is designed so that the target nucleicacid has at least one C (cytosine) or G (guanine) in its base sequenceas a first to third base from its terminal base upon hybridization ofthe probe in the end region with the target nucleic acid.

Preferably, the probe has a base sequence designed so that plural basepairs of the probe-nucleic acid hybrid in the region labeled with thedonor dye form at least one pair between G (guanine) and C (cytosine)upon hybridization of the nucleic acid probe to the target nucleic acid.

More preferably, the probe is labeled with the donor dye at a G(guanine) or C (cytosine) base, at a phosphate group of a nucleotidehaving a G (guanine) or C (cytosine) base or at a OH group of the ribosemoiety of the nucleotide.

When the nucleic acid probe is labeled with the donor dye at a base,phosphate moiety or ribose or deoxyribose moiety in a 5′ end region, thenucleic acid probe may be labeled with the acceptor dye in its chain(strand) or in a 3′ end region inclusive of the 3′ terminal base. Whenthe nucleic acid probe is labeled with the donor dye at a base,phosphate moiety or ribose moiety in a 3′ end region, the nucleic acidprobe may be labeled with the acceptor dye in its strand or in a 5′ endregion inclusive of the 5′ terminal base. The end region can be labeledwith the two dyes. For example, the nucleic acid probe may be labeledwith one of the two dyes at the phosphate moiety and with the other atthe ribose moiety or base moiety, when the base distance between baseslabeled with the donor dye and those labeled with acceptor dye is zeroas described later. Alternatively, the two dyes can be combined with onespacer having a side chain. It is also acceptable in the presentinvention that the nucleic acid probe is labeled with the donor dye andacceptor dye in its chain, as long as the resulting nucleic acid probesatisfies the above conditions.

The fluorescent dyes are preferably combined with the probe at a OHgroup or at an amino group with the interposition of a spacer.

The base distance between bases labeled with the donor dye and baseslabeled with acceptor dye basically depends on the types of the pair ofthe donor dye and acceptor dye and is generally of from 0 to 50 bases,preferably of from 0 to 40 bases, more preferably of from 0 to 35 bases,and typically preferably of from 0 to 15 bases. If it exceeds of 50bases, FRET becomes unstable. In some cases labeling at the distance of15 to 50 bases, the donor dye exhibits an increased fluorescenceintensity although the acceptor dye exhibits a decreased fluorescenceintensity. In other words, the conformation of the nucleic acid probechanges upon hybridization with the target nucleic acid, thus invitingFRET between dyes of the nucleic acid probe to disappear in some cases.The fluorescence intensity of the donor dye increases or decreases afterhybridization, which increasing or decreasing depends on the balancebetween quenching activity of the acceptor dye to the donor dye and thequenching activity of the G-C hydrogen bond complex to the donor dye.The fluorescence intensity decreases when the quenching activity of theG-C hydrogen bond complex is higher than that of the acceptor dye, andincreases when the quenching activity of the G-C hydrogen bond complexis lower than that of the acceptor dye. If the distance is of less than15 bases, both the fluorescence intensities of the donor dye andacceptor dye decrease after hybridization. In this case, the degree ofdecrease does not depend on the conformation of the nucleic acid probeafter hybridization but only on the concentrations of the target nucleicacids in the assay system. To decrease both the fluorescence intensitiesof the donor dye and acceptor dye after hybridization, the distance istypically preferably of 0 to 10 bases.

A typically preferred embodiment of the probe of the present inventionis a nucleic acid probe labeled with the donor dye and acceptor dye atthe above specified distance, in which the donor dye is the BODIPY FLseries dyes, BODIPY 493/503, 5-FAM, Tetramethylrhodamine, or 6-TAMRA,and the acceptor dye is BODIPY 581/591 or X-rhodamine, and the 5′terminal base is G or C and is labeled with the donor dye, or the 3′terminal base is G or C and is labeled with the donor dye.

The novel nucleic acid probe for nucleic acid determination of thepresent invention has the above-mentioned configuration. By thisconfiguration, energy of the excited donor dye transfers to the acceptordye before hybridization with a target nucleic acid. The acceptor dyethereby emits fluorescence with some intensity, and the donor dye isretarded from fluorescence emission and thus exhibits a low level offluorescence intensity. When the nucleic acid probe hybridizes with thetarget nucleic acid, the energy of the donor dye transfers to the G-Chydrogen bond complex formed in the probe-nucleic acid hybrid complex.In addition, FRET disappears due to change in conformation of thenucleic acid probe in some cases. The fluorescent emission of theacceptor dye thus decreases.

The oligonucleotide moiety of the nucleic acid probe of the presentinvention can be prepared according to a conventional production processfor praparing regular oligonucleotides. For example, it can be preparedby a chemical synthetic process or a production process usingmicroorganisms and plasmid vectors or phage vectors (Tetrahedron letters22, 1859-1862 (1981); and Nucleic Acids Research, 14, 6227-6245 (1986)).Commercially available nucleic acid synthesizers, such as ABI 394 (aproduct of PerkinElmer, Inc., USA), are preferably used.

To label the oligonucleotide with the fluorescent dyes, a desired one ofconventionally known labeling method can be used (Nature Biotechnology,14, 303-308 (1996); Applied and Environmental Microbiology, 63,1143-1147 (1997); Nucleic Acids Research, 24, 4532-4535 (1996)). Forexample, the fluorescent dye molecule is combined with theoligonucleotide at the 5′ end in the following manner. Initially, aspacer such as —(CH₂)_(n)—SH is introduced into the phosphate group atthe 5′ end according to conventional methods. Spacer-introduced productsare commercially available and can be used herein (Midland CertifiedReagent Company). In this case, n is from 3 to 8, and preferably 6. Theoligonucleotide can be labeled by combining a fluorescent dye or itsderivative having reactivity with SH group with the spacer. The preparedoligonucleotide labeled with the fluorescent dye can be purified by, forexample, a reversed phase chromatographic method or the like and therebyyields a nucleic acid probe for use in the present invention.

The 3′ terminal base of the oligonucleotide can also be labeled in thefollowing manner.

In this labeling, a spacer, for example, —(CH₂)_(n)—NH₂ is introducedonto an OH group on the C carbon at the 3′ position of the ribose ordeoxyribose. Such spacer-introduced products are also commerciallyavailable and can be used herein (Midland Certified Reagent Company). Inthe above-mentioned example, n is from 3 to 8, and preferably from 4 to7. By reacting an SH— or NH₂-reactive fluorescent dye to the linker orspacer, a labeled oligonucleotide can be prepared.

A base in the chain of the oligonucleotide can also be labeled.

In this labeling, the amino group or OH group of the base may be labeledwith the dye according to the present invention in the same manner as inthe 5′ end or 3′ end (ANALYTICAL BIOCHEMISTRY 225, 32-38 (1998)).

To introduce an amino group, kit reagents such as Uni-link aminomodifier(a product of CLONTECH, USA), FluoReporter Kit F-6082, F-6083, F-6084,and F-10220 (products of Molecular Probes Inc., USA) can beadvantageously used. The fluorescent dye molecule can be jointed to theoligoribonucleotide according to a conventional method.

The labeled oligonucleotide as prepared as above is purified by, forexample, a reversed phase chromatographic method and thereby yields thenucleic acid probe for use in the present invention.

The nucleic acid probe of the present invention can be prepared asdescribed in the above manner. When the prepared nucleic acid probe foruse in the nucleic acid determination of the present inventionhybridizes with the target nucleic acid and forms a probe-nucleic acidhybrid complex, the complex emits fluorescence at a markedly decreasedintensity as compared with that before the formation of the complex.

According to the present invention, one donor dye can be used incombination with plural types of acceptor dyes to thereby yield nucleicacid probes in the number of type(s) equal to that of the combinations.In addition, the nucleic acid probes have the above properties.

In actual nucleic acid determination, decrease in fluorescence intensityof the acceptor dye is measured. Accordingly, one or more types ofnucleic acid probes can be used in parallel at one excitationwavelength. This means that, when one assay system includes one or moretypes of target nucleic acids, addition of such nucleic acid probestogether to the assay system enables determination of the one or moretypes of target nucleic acids in parallel. Specifically, one or moretypes of nucleic acids can be determined in parallel using a simpleapparatus.

A second invention of the present invention is a method for determiningtarget nucleic acids using the above-prepared nucleic acid probes.

The method comprises the steps of adding one or more types of thenucleic acid probes, which probes are ones for use in the nucleic aciddetermination of the present invention, belonging to at least any one ofthe groups with the numerical symbols (the section nos.) 1 to 10 in theafore-mentioned description into an assay system containing one or moretypes of target nucleic acids, the nucleic acid probe(s) being capableof hybridizing with the target nucleic acid(s), being in the number oftype(s) equal to or larger than that of types of the target nucleicacid(s), and emitting fluorescence in different colors; allowing thenucleic acid probe(s) to hybridize to the target nucleic acid(s); anddetermining differential decrease of fluorescence intensities betweenbefore and after hybridization at wavelengths in the number of type(s)equal to or larger than that of types of the nucleic acid probes. Thepresent invention also relates to an assay kit therefor, a data analysismethod for the determination, an device for the determination, and acomputer-readable recording medium with various procedures of a dataanalysis method recorded as a program.

The term “assay system comprising one or more types of target nucleicacids” as used in the present invention means that the assay systemcontains one or plural types of target nucleic acids.

Further, the term “nucleic acid probe for use in the nucleic aciddetermination of the present invention belonging to at least any one ofthe groups with the numerical symbols (the section nos.) 1 to 10 in theafore mentioned description” means as follows.

When the assay system as used herein comprises one type of targetnucleic acid, the type of the nucleic acid probes for nucleic aciddetermination to be added into the assay system may be singular orplural. The term “to may be plural” means that plural types of nucleicacid probes for use in the nucleic acid determination can be used withrespect to the one type of target nucleic acid because the plural typesof the nucleic acid probes can hybridize with plural specific siteshaving different base sequences.

When the assay system contains plural types of target nucleic acids, thepresent invention is characterized by the use of at least plural typesof nucleic acid probes for use in the nucleic acid determination. Thenumber of type(s) of the nucleic acids is at least equal to or largerthan that of the types of the target nucleic acids. The term “to be atleast equal to or larger than that of the types of the target nucleicacids” means that, in one type of target nucleic acid, plural types ofbase sequence sites may be set, which sites are capable of hybridizingindependently with plural types of nucleic acid probes, and plural typesof nucleic acid probes for use in the nucleic acid determination may beadded with respect to one target nucleic acid into the assay system.

The term “plural types of nucleic acid probes for use in the nucleicacid determination probes” in the assay system containing one or pluraltypes of target nucleic acids means as follows. (1) They are pluraldifferent types of probes among the nucleic acid probes for use in thenucleic acid determination according to any one of the groups with thenumerical symbols (the section nos.) 1 to 10 in the above mentioneddescription. Illustrative such probes are plural probes having the samedimensions and structure but being labeled with different dyes.Alternatively, (2) they are plural types of the probes for use in thenucleic acid determination, having different dimensions or structures,being labeled with different dyes and being different in a combinationof those. Namely, the probes may be plural types of probes belonging todifferent groups with the numerical symbols (the section nos.) 1 to 10in the aforementioned description. An example of such a combination ofprobes is a combination of a probe belonging to the group with thenumerical symbol (the section no.) 5, a probe belonging the group withthe numerical symbol(the section no.) 6, and a probe belonging to thegroup with the numerical 10, each of which being labeled with adifferent dye from one another. However, this example illustration doesnot impose any limit on the scope of the present invention.

The terms in the following description have also the same meanings asdefined above.

A third invention of the present invention is a method for analyzing ordetermining polymorphism and/or mutation of one or more types of targetnucleic acids in an assay system, the method including the steps ofadding one or more types of the nucleic acid probes belonging to atleast one of the groups with the numerical symbols (the section nos.) 1to 10 in the above mentioned description into an assay system containingone or more types of target nucleic acids, the nucleic acid probe(s)being capable of hybridizing with the target nucleic acid(s), being inthe number of type(s) equal to or larger than that of the target nucleicacid(s), and being different in colors of emitting fluorescence;allowing the nucleic acid probe(s) to hybridize to the target nucleicacid(s); and determining differential decrease(s) in fluorescenceintensity between before and after hybridization at wavelengths in thenumber of type(s) equal to or larger than that of the types of thenucleic acid probes. The invention also relates to an assay kit for themethod, a data analysis method for the determination, a determinationapparatus, and a computer-readable recording medium with variousprocedures of data analysis recorded as a program.

Namely, the nucleic acid probes of the present invention can beadvantageously utilyzed not only for the nucleic acid determination butalso for a method for analyzing or determining polymorphism and/ormutation of a target nucleic acid.

Specifically, the present invention provides a convenient method byusing it in combination with the following DNA chip (Protein, NucleicAcid and Enzyme, 43, 2004-2011 (1998)). The fluorescence intensityvaries depending on whether a C-G pair is formed or not uponhybridization of the nucleic acid probe of the present invention. Byallowing the nucleic acid probe of the present invention to hybridize tothe target nucleic acid and determining the fluorescent intensity,polymorphism and/or mutation of the target nucleic acid can be analyzedor determined. Specific methods will be described in examples later. Thetarget nucleic acid in these method may be an amplified productamplified according to desired one of various nucleic acid amplificationmethods or may be an extracted product. Further, the type of the targetnucleic acid is not specifically limited, as long as it has a guaninebase or cytosine base in its strand or at its end. If the target nucleicacid does not have a guanine base or cytosine base in its strand or atits end, the fluorescence intensity does not decrease. Accordingly, themethod of the present invention can analyze or determine G→A, G←A, C→T,C←T, G→C, G←C mutations or substitutions, i.e.; single nucleotidepolymorphisms (SNPs) and other polymorphisms. Such polymorphisms havebeen analyzed by sequencing the target nucleic acid using theMaxam-Gilbert method or dideoxy method.

The nucleic acid probe of the present invention was included in an assaykit for analyzing or determining polymorphism and mutation of a targetnucleic acid, the resulting kit can be advantageously used as an assaykit for analyzing or determining polymorphism and/or mutation of thetarget nucleic acid.

On the analysis of data obtained by the method for analyzing ordetermining polymorphism and/or mutation of a target nucleic acid of thepresent invention, a processing step may be added to correct thefluorescence intensity, which is emitted from the reaction system whenthe target nucleic acid is hybridized with the nucleic acid probe of thepresent invention by the intensity of fluorescence emitted from thereaction system when the target nucleic acid and the nucleic acid probeare not hybridized with each other. The data so processed are providedwith high reliability.

Accordingly, the present invention also provides a data analysis methodfor the method which analyzes or measures polymorphism and/or mutationof a target nucleic acid.

The present invention also features a system for analyzing ordetermining polymorphism and/or mutation of a target nucleic acid, whichhas processing means for correcting a fluorescence intensity of areaction system, in which the target nucleic acid is hybridized with thenucleic acid probe according to the present invention, in accordancewith a fluorescence intensity of the reaction system in which the targetnucleic acid is not hybridized with the nucleic acid probe according tothe present invention.

The present invention further features a computer-readable recordingmedium with procedures recorded as a program therein for making acomputer perform a processing step in which, when analyzing dataobtained by the method for analyzing or determining polymorphism and/ormutation of a target nucleic acid, a fluorescence intensity of areaction system, in which the target nucleic acid is hybridized with thenucleic acid probe according to the present invention, is corrected inaccordance with a fluorescence intensity of the reaction system in whichthe target nucleic acid or gene is not hybridized with the nucleic acidprobe according to the present invention.

The third invention further provides a device for determiningconcentrations of plural nucleic acids, which comprises plural pieces ofthe nucleic acid probes for nucleic acid determination of the presentinvention combined with the surface of a substrate to allow targetnucleic acids to hybridize with the probes to thereby determine thetarget nucleic acids. The device may constitute a device (a chip) fordetermining plural nucleic acids, which comprises the nucleic acidprobes arranged and immobilized in an array on the surface of a solidsubstrate. The device may further comprise at least one each temperaturesensor and heater per probe, which are disposed on a surface at an areaof the solid substrate opposite to the nucleic acid probe. In thisdevice, the temperatures of regions carrying the nucleic acid probes canbe controlled to attain the optimum temperatures.

The nucleic acid probe according to the present invention may beimmobilized on a surface of a solid (support layer), for example, on asurface of a slide glass. In this case, the probe may preferably beimmobilized on the end not labeled with the fluorescent dye. The probeof this form is now called a “DNA chip”. These DNA chips can be used formonitoring gene expressions, determining base sequences, analyzingmutations or analyzing polymorphisms such as single nucleotidepolymorphism (SNP). Needless to say, they can also be used as devices(chips) for determining nucleic acids.

To bind the nucleic acid probe of the present invention, for example, toa surface of slide glass, the slide glass coated with polycations suchas polylysine, polyethyleneimine and polyalkylamine, a slide glasscarrying aldehyde groups introduced thereon or a slide glass carryingamino groups introduced thereon is initially prepared. Subsequently thenucleic acid probe can be immobilized to the slide glass in thefollowing manner. i) Phosphate groups of the probe are allowed to reactwith the slide glass coated with polycations; ii) a probe carryingintroduced amino groups is allowed to react with the slide glasscarrying aldehyde groups; and iii) a probe carrying introducedpyridinium dichromate (PDC), amino groups or aldehyde groups is allowedto react with the slide glass carrying amino groups (Fodor, P. A., etal., Science, 251, 767-773, 1991; Schena, W., et al., Proc. Natl. Acad.Sci., U.S.A., 93, 10614-10619, 1996; McGal, G., et al., Proc. Natl.Acad. Sci., U.S.A., 93, 13555-13560, 1996; Blanchad, A. P., et al.,Biosens. Bioelectron., 11, 687-690, 1996).

A device comprising the nucleic acid probes for nucleic aciddetermination of the present invention arranged and bound in an array onthe surface of a solid support permits more convenient determination ofthe target nucleic acids.

One or more types of target nucleic acids can be determined in parallelby using a device comprising multiple types of the nucleic acid probesof the present invention having different base sequences arranged andbound on the surface of one solid support.

The device preferably further comprises at least one each temperaturesensor and heater per probe, which are disposed on a surface opposite tothe nucleic acid probes. In this device, the temperatures of regionscarrying the nucleic acid probes can be controlled to thereby attain theoptimum temperatures.

The determination method using the device of the present invention canbe basically performed only by placing a solution containing a targetnucleic acid on the surface of the solid support on which the nucleicacid probes are bound and then to thereby allow the probes to hybridizewith the target nucleic acid. As a result, a change in the intensity offluorescence takes place corresponding to the amount of the targetnucleic acid, and the target nucleic acid can be then determined basedon the changes in fluorescence. Further, binding multiple types of thenucleic acid probes of the present invention having different basesequences to the surface of a single support makes it possible todetermine in parallel the concentrations of multiple types of targetnucleic acids. The device is therefore a novel DNA chip, since it can beused in determination of target nucleic acids in the same use as inconventional DNA chips. Nucleic acids other than the target nucleicacids do not change the fluorescence emission under optimum reactionconditions, and the device does not require procedures for washing outunreacted nucleic acids. In addition, when the device comprisesmicroheaters that can control the temperature per nucleic acid probe ofthe present invention to achieve the optimum reaction condition of eachprobe, the concentrations can be determined more precisely. The devicecan analyze dissociation curves between the nucleic acid probes of thepresent invention and the target nucleic acids by continuously changingthe temperatures using the microheaters and determining the intensity offluorescence during the changing of the temperature. It can also analyzethe properties of the hybridized nucleic acids and/or detect SNPs basedon differences in the dissociation curves.

In a conventional device for determining the concentration of a targetnucleic acid, nucleic acid probes not modified with any fluorescent dyeare bound or fixed to the surface of a solid support and subsequent tohybridization with the nucleic acid probes labeled with a fluorescentdye, an unhybridized target nucleic acids are washed out, followed bythe measurement of intensity of fluorescence from the remainingfluorescent dye

To label the target nucleic acid with the fluorescent dye, the followingsteps can be followed, for example, when specific mRNA is chosen as atarget nucleic acid: (1) mRNAs is extracted in its entirety from cells;and (2) cDNAs is synthesized using a reverse transcriptase whileinserting a nucleoside modified with a fluorescent dye. The presentinvention does not require these operations.

A number of various probes are applied in spots on the device. Optimumhybridization conditions, for example, temperatures or the like, fornucleic acids to be hybridized to the individual probes are differentfrom each other. Theoretically speaking, it is therefore necessary toconduct a hybridization reaction and a washing operation under optimalconditions for each probe (at each spot). This is however physicallyimpossible. For all the probes, hybridization is conducted at the sametemperature and further, washing is also carried out at the sametemperature with the same washing solution. The device is, therefore,accompanied by a drawback that a nucleic acid does not hybridizealthough its hybridization is desired or that, even if its hybridizationtakes place, the nucleic acid is readily washed off as the hybridizationis not strong. For these reasons, the accuracy of quantification of thenucleic acid is low. The present invention does not have such a drawbackbecause the above-mentioned washing operation is not needed. Further, ahybridization reaction can be conducted at an optimal temperature foreach probe of the present invention by independently arranging amicroheater at the bottom of each spot and controlling the hybridizationtemperature. Accordingly, the accuracy of quantification has beensignificantly improved in the present invention.

According to the present invention, one or more types of target nucleicacids in an assay system can be easily and specifically determined in ashort time by using the nucleic acid probes or device for nucleic aciddetermination of the present invention. The determination method will bedescribed below.

In the determination method of the present invention, the nucleic acidprobes for nucleic acid determination of the present invention in thenumber of type(s) equal to or larger than that of target nucleic acidsare added to the assay system to allow the probes to hybridize with thetarget nucleic acids according to a conventional or known procedure(Analytical Biochemistry, 183, 231-244 (1989); Nature Biotechnology, 14,303-308 (1996); Applied and Environmental Microbiology, 63, 1143-1147(1997)). For example, hybridization is performed at a salt concentrationof 0 to 2 M and preferably 0.1 to 1.0 M, and at pH 6 to 8 and preferablypH 6.5 to 7.5.

The reaction temperature preferably falls within the range of Tm±10° C.,where Tm is the melting temperature of a nucleic acid hybrid complexformed as a result of hybridization of the nucleic acid probe of thepresent invention with a specific site of the target nucleic acid. Bysetting the reaction temperature within this range, non-specifichybridization can be avoided. If the reaction temperature is lower than[Tm−10° C.], non-specific hybridization may occur, and if it exceeds[Tm+10° C.], hybridization may not occur. Tm can be determined in thesame manner as in an experiment to design the nucleic acid probes of thepresent invention. Specifically, an oligonucleotide that is capable ofhybridizing with the nucleic acid probe of the present invention and hasa complementary base sequence to the nucleic acid probe is chemicallysynthesized using, for example, the nucleic acid synthesizer, and Tm ofa nucleic acid hybrid complex with the nucleic acid probe is determinedaccording to a conventional procedure. When the assay system containsplural target nucleic acids, the average of Tms of plural nucleic acidhybrid complexes or Tm of a target nucleic acid most valued can be usedas Tm.

The reaction time is from 1 second to 180 minutes, and preferably from 5seconds to 90 minutes. If the reaction time is less than 1 second, anincreasing amount of the nucleic acid probes of the present inventionmay remain unreacted. In contrast, an excessively long reaction time isuseless. The reaction time significantly depends on the types of thenucleic acids, i.e., the lengths or base sequences of nucleic acids.

Thus, the nucleic acid probes for nucleic acid determination of thepresent invention are allowed to hybridize with the target nucleicacids. Next, the fluorescence intensity of the hybridization reactionsystem before and after hybridization is measured at wavelengths in thenumber of type(s) equal to or larger than that of the types of the usednucleic acid determination probes using a fluorophotometer, and decreaseat each wavelength is determined by calculation. The magnitudes ofdecrease are proportional to the concentrations of the target nucleicacids, and thereby the concentrations of the target nucleic acids can bedetermined based on the decrease.

The concentrations of the target nucleic acids in the reaction mixtureare preferably from 0.1 to 10.0 nM. The concentrations of the nucleicacid probes of the present invention in the reaction mixture arepreferably 1.0 to 25.0 nM. To plot a calibration curve, the ratio of thenucleic acid probe of the present invention to the target nucleic acidis preferably from 1.0 to 2.5.

Initially, a calibration curve is plotted under the above conditions toactually determine a target nucleic acid in an unknown concentration ina sample. When plural target nucleic acids are to be determined,calibration curves are plotted at individual measuring wavelengths ofthe nucleic acid probes of the present invention corresponding to thetarget nucleic acids. The nucleic acid probes of the present inventionin plural concentrations are added to the sample, and decreases influorescence intensity of the individual nucleic acid probes aredetermined. In this procedure, a preferred probe concentration isdefined as the concentration corresponding to the maximum decrease ofthe measured fluorescence intensities. A decrease in fluorescenceintensity is determined using the probe in the preferred concentration,and the amount of the target nucleic acid is read from the decreaseplotted in the calibration curve.

A fourth invention of the present invention is applied to nucleic aciddetermination processes such as fluorescence in situ hybridization(FISH), polymerase chain reactions (PCRs), ligase chain reactions(LCRs), SD process, competitive hybridization, and transcription-basedamplification system (TAS).

Embodiments of the invention will be described below.

a) Application to FISH

The method of the present invention can be applied to nucleic acidscontained in cells of microorganisms, plants or animals or thosecontained in homogenates of the respective cells. The method of thepresent invention can also be suitably applied to nucleic acids in cellsof a cultivation system of microorganisms (e.g., a co-cultivation systemof microorganisms or a symbiotic cultivation system of microorganisms),in which various kinds of microorganisms are contained together or amicroorganism and other animal- or plant-derived cells are containedtogether and cannot be isolated from each other, or in a homogenate orthe like of the cells of the cultivation system.

The term “microorganisms” as used herein means microorganisms in generalsense, and no particular limitation is imposed thereon. Examples of suchmicroorganisms can include eukaryotic microorganisms and prokaryoticmicroorganisms, and also mycoplasmas, virus and rickettsias. The term “atarget nucleic acid” as used in connection with such a microorganismsystem means a nucleic acid with a base sequence specific to cells of acell strain which is desired to be investigated, for example, as to howit is acting in the microorganism strain. Illustrative examples caninclude 5S rRNAs, 16S rRNAs and 23S rRNAs of certain specific cellstrains and particular sequences of their gene DNAs.

According to the present invention, a nucleic acid probe is added to aco-cultivation system of microorganisms or a symbiotic cultivationsystem of microorganisms and the amount of 5S rRNA, 16S rRNA or 23S rRNAof a particular cell strain or its gene DNA, thereby making it possibleto determine the viable count of the particular strain in the system.Incidentally, a viable count of a particular cell strain in aco-cultivation system of microorganisms or a symbiotic cultivationsystem of microorganisms can be determined by adding the nucleic acidprobe to a homogenate of the system and then measuring the intensity offluorescence emission from the fluorescent dye before and afterhybridization. It is to be noted that this method also falls within thetechnical scope of the present invention.

The above-described determination method can be carried out as will bedescribed hereinafter. Before the addition of the nucleic acid probe ofthe present invention, the temperature, salt concentration and pH of theco-cultivation system of microorganisms or the symbiotic cultivationsystem of microorganisms are adjusted to meet the conditions describedabove. It is also preferable to adjust the concentration of the specificcell strain, which is contained in the co-cultivation system ofmicroorganisms or the symbiotic cultivation system of microorganisms, to10⁷ to 10¹² cells/mL, preferably 10⁹ to 10¹⁰ cells/mL in terms of viablecount. These adjustments can be achieved by dilution, centrifugal orlike concentration, or the like. A viable count smaller than 10⁷cells/mL results in low fluorescence intensity and larger determinationerror. A viable count larger than 10¹² cells/mL, on the other hand,leads to excessively high fluorescence intensity from the co-cultivationsystem of a microorganism or the symbiotic cultivation system ofmicroorganisms, so that the viable count of the particular microorganismcannot be determined quantitatively. However, this range depends uponthe performance of a fluorimeter to be used.

The concentration of the nucleic acid probe of the present invention tobe added depends upon the viable count of the particular cell strain inthe co-cultivation system of microorganisms or the symbiotic cultivationsystem of microorganisms and, at a viable count of 10⁸ cells/mL, may bein a range of from 0.1 to 10.0 nM, preferably in a range of from 0.5 to5 nM, more preferably 1.0 nM. A probe concentration lower than 0.1 nMcannot provide any data which accurately reflects the viable count ofthe particular microorganism. The optimal concentration of the nucleicacid probe according to the present invention, however, cannot bespecified in any wholesale manner because it depends upon theconcentration of a target nucleic acid in cells.

Upon hybridizing the nucleic acid probe to the 5S rRNA, 16S rRNA or 23SrRNA of the particular cell strain or its gene DNA in the presentinvention, the reaction temperature may be set as described above.Further, the hybridization time may also be set as described above.

The nucleic acid probe according to the present invention is hybridizedto the 5S rRNA, 16S rRNA or 23S rRNA of the particular cell strain orits gene DNA under such conditions as described above. Intensities offluorescence from the fluorescent dye in the co-cultivation system ofmicroorganisms or the symbiotic cultivation system of microorganismsbefore and after the hybridization are then measured.

The degree of the above determined increase in fluorescent intensity isproportional to the number of specified microorganism(s); its number isproportional to amounts of 5SrRNA, 16SrRNA, 23SrRNA or the genes ofthose contained in cells.

In the present invention, no particular limitation is imposed oncomponents other than the microorganisms in the co-cultivation system ofmicroorganisms or the symbiotic cultivation system of microorganisms,insofar as the components do not interfere with the hybridizationbetween the nucleic acid probe according to the present invention andthe 5S rRNA, 16S rRNA or 23S rRNA of the particular cell strain or itsgene DNA and further, do not inhibit the emission of fluorescence fromthe fluorescent dye or the action of the quencher substance labeled onthe oligonucleotide. For example, phosphates such as KH₂PO₄, K₂HPO₄,NaH₂PO₄ and Na₂HPO₄, inorganic nitrogens such as ammonium sulfate,ammonium nitrate and urea, various salts of ions such as magnesium,sodium, potassium and calcium, various salts such as the sulfates,hydrochlorides, carbonates and the like of trace metal ions such asmanganese, zinc, iron and cobalt, and vitamins may be contained toadequate extent. If the above-described interference or inhibition isobserved, it may be necessary to separate cells of the pluralmicroorganisms from the cultivation system by an operation such ascentrifugal separation and then to resuspend them in a buffer or thelike.

Usable examples of the buffer can include various buffers such asphosphate buffer, carbonate buffer, Tris-HCl buffer, Tris-glycinebuffer, citrate buffer, and Good's buffer. The buffer should be adjustedto a concentration not inhibiting the hybridization or the emission offluorescence from the fluorescent dye. This concentration depends uponthe kind of the buffer. The pH of the buffer may range from 4 to 12,with 5 to 9 being preferred.

b) Application to PCR Methods

The present invention can be applied to any method insofar as it is aPCR method. A description will hereinafter be made of an application ofthe present invention to a real-time quantitative PCR method.

In the real-time quantitative PCR method, PCR is conducted using aspecific nucleic acid probe according to the present invention, and adecrease in fluorescent intensity of a reaction system after a reactionrelative to fluorescent intensity of a reaction system before thereaction is determined in real time.

The term “PCR” as used herein means a variety of PCR methods. Examplescan include RT-PCR, RNA-primed PCR, stretch PCR, reverse PCR, PCR makinguse of an Alu sequence, multiple PCR, PCR making use of a mixed primer,and PCR making use of PNA. Further, the term “quantitative” means, inaddition to quantitation in general sense, quantitation of such anextent as detection as described above.

As described above, the term “target nucleic acid” as used herein meansa nucleic acid the existing amount of which is intended to bedetermined, irrespective whether it is in a purified form or not andfurther irrespective of its concentration. Various other nucleic acidsmay also exist. For example, the target nucleic acid may be a specificnucleic acid in a co-cultivation system microorganisms (a mixed systemof RNAs or gene DNAs of plural microorganisms) or a symbioticcultivation system of microorganisms (a mixed system of RNAs or geneDNAs of plural animals, plants and/or microorganisms), the amplificationof which is intended. Purification of the target nucleic acid, ifneeded, can be conducted by a method known per se in the art. Forexample, purification can be effected using a purification kit or thelike available on the market.

The conventionally-known quantitative PCR methods individually amplify,in the presence of Mg ions, a target nucleic acid (DNA or RNA) by usingDATP, dGTP, dCTP, dTTP or dUTP, the target nucleic acid, Taq polymerase,a primer, and a nucleic acid labeled with a fluorescent dye or anintercalator while repeatedly changing the temperature between low andhigh levels, and monitor increases in fluorescence emission from thefluorescent dye in real time in the course of the amplification [JikkenIgaku (Laboratory Medicine), 15(7), 46-51, Yodosha (1997)].

On the other hand, the quantitative PCR method according to the presentinvention is characterized in that one or more types of the targetnucleic acids are amplified by using the nucleic probe of the presentinvention of the number of type(s) equal to or larger than that of thetarget nucleic acids and a change in fluorescent intensity in a reactionsystem is determined using wavelengths of the number of type(s) equal toor larger than that of the nucleic acid probes. The number of base(s) ina preferred probe of the present invention for use in the quantitativePCR according to the present invention may be from 5 to 50, preferablyfrom 10 to 25, notably from 15 to 20. No particular limitation isimposed on the probe insofar as it hybridizes to amplification productsof the target nucleic acid in PCR cycles. The probe may be designed ineither a forward type or a reverse type.

Preferred nucleic acid probes for use herein are described below:

(1) Preferred nucleic acid probes are designed so that plural base pairsof the probe-nucleic acid hybrid at the region labeled with the donordye constitute at least one G (guanine) and C (cytosine) pair upon orafter hybridization with the target nucleic acids.

More preferably,

(2) in the nucleic acid probes (1), more preferred are those labeledwith the donor dye at a G (guanine) or C (cytosine) base, at a phosphategroup of a nucleotide having a G or C base, or at a OH group of a riboseor deoxyribose and labeled with the acceptor dye in a 3′ end regionexclusive of the 31 end or in a chain.

(3) In the nucleic acid probes (2), more preferred are those labeledwith the acceptor dye in a chain in the vicinity of a region labeledwith the donor dye.

Specifically, among the nucleic acid probes of the present invention, aprobe is utilized as a primer, which probe belongs to the above (1) andis labeled with a donor dye at a base of the 5′ end domain or the baseof the 5′ end of a olygonucleotide of the nucleic acid probe and with anacceptor dye at the base in a strand(chain) of the olygonucleotide or atthe base of its 3′ end.

When the nucleic acid probe of the present invention is used as a primerand cannot be designed to have G or C at the 3′ end or 5′ end due to thebase sequence of the target nucleic acid, the objects of the presentinvention can also be achieved by adding 5′-guanylic acid or guanosine,or 5′-cytidylic acid or cytidine to the 5′ end of an oligonucleotide asa primer designed based on the base sequence of the target nucleic acid.The objects of the present invention can also be advantageously achievedby adding 5′-guanylic acid or 5′-cytidylic acid to the 3′ end. The term“nucleic acid probes designed so that to have G or C at 3′ end or 5′end” used herein means and includes probes designed based on the basesequences of target nucleic acids, as well as those prepared by adding5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine to the 5′end of the designed probes and those prepared by adding 5′-guanylic acidor 5′-cytidylic acid to the 3′ end of the designed probes.

In particular, when a nucleic acid probe among the above-described probe(2) of the present invention is labeled with a donor dye at a base ofthe 3′ end domain (including the base of the 3′ end) or ribose ordeoxyribose of the 3′ end, such a probe and with an acceptor dye at abase in the strand is designed to be not used as a primer. The methodusing such a single probe of the present invention can be utilized asone substituting for a conventional method using two(fluorescent-dye-labeled) probes that have been up to the present usedin a real-time quantitative PCR method utilizing the FRET phenomenon.The probe is added to a PCR reaction system, and PCR is then conducted.During a nucleic acid extending reaction, the probe which has been in aform hybridized with the target nucleic acid or amplified target nucleicacid is degraded by polymerase and is dissociated off from the nucleicacid. hybrid complex. The intensity of fluorescence of the reactionsystem at this time or the reaction system in which a nucleic aciddenaturing reaction has completed is measured. Further, the intensity offluorescence of the reaction system in which the target nucleic acid oramplified target nucleic acid has hybridized with the probe (i.e., thereaction system at the time of an annealing reaction or at the time ofthe nucleic acid extending reaction until the probe is eliminated fromthe nucleic acid hybrid complex by polymerase). By calculating the rateof a decrease of the latter fluorescence intensity from the formerfluorescence intensity, the amplified nucleic acid is determined. Theintensity of fluorescence is high when the probe has completelydissociated from the target nucleic acid or amplified target nucleicacid by the nucleic acid denaturing reaction or when the probe has beendegraded out from the hybrid complex of the probe and the target nucleicacid or amplified nucleic acid at the time of extension of the nucleicacid. However, the intensity of fluorescence of the reaction system inwhich an annealing reaction has been completed and the probe has fullyhybridized to the target nucleic acid or amplified target nucleic acidor of the reaction system until the probe is degraded out of the hybridcomplex of the probe and the target nucleic acid or amplified targetnucleic acid by polymerase at the time of a nucleic acid extendingreaction is lower than the former. The decrease in the intensity offluorescence is proportional to the concentration of the amplifiednucleic acid.

In this case, the base sequence of the probe (2) may desirably bedesigned such that the Tm of a nucleic acid hybrid complex, which isavailable upon hybridization of the probe with the target nucleic acid,falls within a range of the Tm value of the nucleic acid hybrid complexas a primer ±15° C., preferably ±5° C. If the Tm of the probe is lowerthan (the Tm value of the primer −5° C.), especially (the Tm value ofthe primer −15° C.), the probe does not hybridize so that no decreasetakes place in the fluorescence emission from the fluorescent dye. Ifthe Tm of the probe is higher than (the Tm value of the primer +5° C.),especially (the Tm value of the primer +15° C.), on the other hand, theprobe also hybridizes to nucleic acid or acids other than the targetnucleic acid so that the specificity of the probe is lost.

The probes other than the probe (2), especially the probe (1) is addedas a primer to PCR reaction systems. Except for the PCR method accordingto the present invention, no PCR method is known to make use of a primerlabeled with a fluorescent dye. As the PCR reaction proceeds, theamplified nucleic acid is progressively labeled with the fluorescent dyeuseful in the practice of the present invention. Accordingly, theintensity of fluorescence of the reaction system in which the nucleicacid denaturing reaction has completed is high but, in the reactionsystem in which the annealing reaction has completed or the nucleic acidextending reaction is proceeding, the intensity of fluorescence of thereaction system is lower than the former intensity of fluorescence.

The PCR reaction can be conducted under similar conditions as inconventional PCR methods. It is, therefore, possible to conductamplification of a target nucleic acid in a reaction system theconcentration of Mg ions in which is low (1 to 2 mM). Needless to say,the present invention can also be conducted even in a reaction system inwhich Mg ions are contained at such a high concentration (2 to 4 mM) asthat employed in the conventionally-known quantitative PCR methods.

Incidentally, in the PCR method according to the present invention, Tmvalue can be determined by conducting the PCR of the present inventionand then analyzing the melting curve of the nucleic acids with respectto the amplification products by using the number of type(s) ofwavelengths equal to that of types of the used nucleic acid probes. Thismethod is a novel analysis method of a melting curve of a nucleic acid.In this method, the nucleic acid probe employed as a nucleic acid probeor primer in the PCR method of the present invention can be usedsuitably.

In this case, designing of the base sequence of the nucleic acid probeaccording to the present invention into a sequence complementary with aregion containing SNP (single nucleotide polymorphism) makes it possibleto detect SNP from a difference, if any, in a dissociation curve of thenucleic acid from the nucleic acid probe of the present invention byanalyzing the dissociation curve after completion of PCR. If a basesequence complementary with an SNP-containing sequence is used as asequence for the nucleic acid probe of the present invention, a Tm valueavailable from a dissociation curve between the sequence of the probeand the SNP-containing sequence becomes higher than a Tm value availablefrom a dissociation curve between the sequence of the probe and theSNP-free sequence.

The fifth invention of the present invention relates to the method foranalyzing data obtained by the above-described real-time quantitativePCR method.

A real-time quantitative PCR method is now practiced in real time by asystem which is composed of a reactor for conducting PCR, equipment fordetecting fluorescence emission from a fluorescent dye, a userinterface, namely, a computer-readable recording medium with variousprocedures of a data analysis method recorded as a program (also called“sequence detection software system”), and a computer for controllingthem and analyzing data. Determination by the present invention is alsoconducted by such a system.

A description will first be made of an analyzer for real-timequantitative PCR. Any system can be used in the present inventioninsofar as it can monitor PCR in real time. Particularly suitableexamples can include “ABI PRISM™ 7700 Sequence Detection System SDS7700” (manufactured by PerkinElmer Applied Biosystems, Inc., U.S.A.) and“LightCycler™ System” (manufactured by Roche Diagnostics, Germany).

The above-described PCR reactor is an apparatus for repeatedlyconducting a thermal denaturing reaction of a target nucleic acid, anannealing reaction and an extending reaction of the nucleic acid (thesereactions can be repeatedly conducted, for example, by successivelychanging the temperature to 95° C., 60° C. and 72° C.). The detectionsystem comprises a fluorescence emitting argon laser, a spectrograph anda CCD camera. Further, the computer-readable recording medium with thevarious procedures of the data analysis method recorded as the programis used by installing it in the computer, and contains a programrecorded therein for controlling the above-described system via thecomputer and also for processing and analyzing data outputted from thedetection system.

The data analysis program recorded in the computer-readable recordingmedium comprises the following steps: measuring the intensity offluorescence cycle by cycle, displaying each measured fluorescenceintensity as a function of cycles, namely, as a PCR amplification ploton a display of the computer, calculating a threshold cycle number (Ct)at which the intensity of fluorescence is begun to be detected, forminga working line useful in determining from Ct values the number ofcopy(ies) of the nucleic acid in the sample, and printing data and plotvalues in the respective steps. When PCR is exponentially proceeding, alinear relationship is established between the logarithm of the numberof copy(ies) of the target nucleic acid at the time of initiation of PCRand Ct. It is therefore possible to calculate the number of copy(ies) ofthe target nucleic acid at the time of initiation of PCR by forming aworking line based on known copy numbers of the target nucleic acid anddetecting the Ct of a sample which contains the target nucleic acid thenumber of copy(ies) of which is unknown. ps c) A Method for AnalyzingData Obtained by the above PCR Method, an Apparatus for Analyzing theData and a Computer-readable Recording Medium with Various ProceduresMaking the Computer Conduct the Method.

The PCR-related invention such as the above-described data analysismethod is a method for analyzing data obtained by such a real-timequantitative PCR method as described above. Its respective features willbe described hereinafter.

A first feature resides in a processing step for correcting afluorescence intensity of a reaction system, which is measured when theamplified nucleic acid hybridizes to a nucleic acid probe according tothe present invention in the method for analyzing data obtained by thereal-time quantitative PCR method, by a fluorescence intensity of thereaction system as obtained when the above-described hybrid complex ofthe nucleic acid probe of the present invention and the target nucleicacid or the nucleic acid hybrid complex has dissociated in each cycle,namely, the first feature resides in a correction-processing step.

As a specific example of “the reaction system . . . when the amplifiedtarget nucleic acid hybridizes to a nucleic acid probe according to thepresent invention”, a reaction system upon conducting a nucleic acidextending reaction or annealing at 40 to 85° C., preferably 50 to 80° C.in each cycle of PCR can be mentioned. It also means a reaction systemin which such a reaction has been completed. The actual temperaturedepends upon the length of the amplified nucleic acid.

Further, “the reaction system . . . when the above-described nucleicacid hybrid complex has dissociated” can be a reaction system uponconducting thermal denaturation of the nucleic acid in each cycle ofPCR, specifically at a reaction temperature of from 90 to 100° C.,preferably 94 to 96° C. Illustrative is a system in which the reactionhas been completed.

Any correction processing can be used as the correction processing inthe correction processing step insofar as it conforms with the objectsof the present invention. Specifically, correction processing includinga processing step by the following formula (1) or formula (2) can beexemplified.f _(n) =f _(hyb,n) /f _(den,n)  (1)f _(n) =f _(den,n) /f _(hyb,n)  (2)where

-   -   f_(n): correction-processed value in an n^(th) cycle as        calculated in accordance with the formula (1) or formula (2),    -   f_(hyb,n): intensity value of fluorescence of the reaction        system available after the amplified nucleic acid has hybridized        to the nucleic acid probe labeled with the fluorescent dye in        the n^(th) cycle, and    -   f_(den,n): intensity value of fluorescence of the reaction        system available after the nucleic acid hybrid complex has        dissociated in the n^(th) cycle.

This step includes a sub-step in which correction-processed valuesobtained by the above-described processing are displayed on a computerdisplay and/or the correction-processed values are likewise displayedand/or printed in the form of a graph as a function of cycles.

A second feature resides in a data analysis method, which comprisesintroducing correction-processed values, which have been calculated inaccordance with the formula (1) or formula (2) in individual cycles,into the following formula (3) or formula (4) to calculate rates orpercentages of changes in fluorescence between samples in the individualcycles:F _(n) =f _(n) /f _(a)  (3)F _(n) =f _(a) /f _(n)  (4)where

-   -   F_(n): rate or percentage of a change in fluorescence in an        n^(th) cycle as calculated in accordance with the formula (3) or        formula (4),    -   f_(n): correction-processed value calculated in the n^(th) cycle        as calculated in accordance with the formula (1) or formula (2),        and    -   f_(a): correction-processed value calculated in a given cycle        before a change in f_(n) is observed as calculated in accordance        with the formula (1) or formula (2), and in general, a        correction-processed value, for example, in one of 10^(th) to        40^(th) cycles, preferably one of 15^(th) to 30^(th) cycles,        more preferably one of 20^(th) to 30^(th) cycles is adopted; and        comparing the rates or percentages of changes in fluorescence.

This step includes a sub-step in which calculated values obtained by theabove-described processing are displayed on a computer display and/orare printed or comparative values or the calculated values are likewisedisplayed and/or printed in the form of a graph as a function of cycles.This sub-step may be applied or may not be applied to thecorrection-processed values obtained by the formula (1) or formula (2).

A third feature resides in a data analysis method, which comprises thefollowing processing steps:

3.1) performing processing in accordance with the following formula (5),(6) or (7) by using data of rates or percentages of changes influorescence as calculated in accordance with said formula (3) or (4):log_(b)(F_(n)), ln(F_(n))  (5)log_(b){(1−F _(n))×A}, ln{(1−F _(n))×A}  (6)log_(b){(F _(n)−1)×A}, ln{(F _(n)−1)×A}  (7)where

-   -   A,b: desired numerical values, preferably integers, more        preferably natural numbers and, when A=100, b=10, {(F_(n)−1)×A}        is expressed in terms of percentage (%), and    -   F_(n): rate or percentage of a change in fluorescence in an        n^(th) cycle as calculated in accordance with the formula (3) or        formula (4),

3.2) determining a cycle in which said processed value of saidprocessing step 3.1) has reached a constant value,

3.3) calculating a relational expression between cycle of a nucleic acidsample of a known concentration and the number of copy(ies) of saidtarget nucleic acid at the time of initiation of a reaction, and

3.4) determining the number of copy(ies) of said target nucleic acid inan unknown sample upon initiation of PCR.

Preferably, these steps are performed in the order of3.1)→3.2)→3.3)→3.4).

Each of these steps 3.1) to 3.3) may include a sub-step in whichprocessed values obtained by the corresponding processing are displayedon a computer display and/or the processed values are likewise displayedand/or printed in the form of a graph as a function of cycles. The step3.4) should include at least a printing sub-step as the processed valuesobtained in the step 3.4) have to be printed, although the processedvalues obtained in the step 3.4) may also displayed on a computerdisplay.

Incidentally, the correction-processed values obtained by the formula(1) or (2) and the calculated values obtained by the formula (3) or (4)may be or may not be displayed on a computer display and/or printed inthe form of graphs as a function of cycles, respectively. Thesedisplaying and/or printing sub-steps may, therefore, be added as needed.

A fourth feature resides in an analysis system for real-timequantitative PCR, which comprises processing and storing means forperforming a data analysis method for the above-described real-timequantitative PCR method of the present invention.

A fifth feature resides in a computer-readable recording medium withindividual procedures of a data analysis method, which is adapted toanalyze PCR by using the analysis system for the real-time quantitativePCR, stored as a program therein, wherein the program is designed tomake a computer perform the individual procedures of the data analysismethod of the present invention.

A sixth feature resides in a novel method for determining a nucleicacid, which comprises using the data analysis method, determinationand/or analysis system and/or recording medium of the present inventionin the nucleic acid determination method.

Specifically, the sixth feature resides in an analysis method, whichcomprises the following steps: gradually heating a nucleic acid, whichhas been amplified by the PCR method of the present invention, from alow temperature until complete denaturation of the nucleic acid (forexample, from 50° C. to 95° C.); measuring an intensity of fluorescenceat short time intervals (for example, at intervals equivalent to atemperature rise of from 0.2° C. to 0.5° C.) during the heating step;displaying results of the measurement as a function of time on adisplay, namely, a melting curve of the nucleic acid; differentiatingthe melting curve to obtain differentiated values (−dF/dT, F: intensityof fluorescence, T: time); displaying the differentiated values asderivatives on the display; and determining a point of inflection fromthe derivatives. In the present invention, the intensity of fluorescenceincreases as the temperature rises. Preferable results can be obtainedin the present invention by adding to the above-described step a furtherprocessing step in which in each cycle, the intensity of fluorescence atthe time of the nucleic acid extending reaction, preferably at the timeof completion of the PCR reaction is divided by the value offluorescence intensity at the time of the thermal denaturing reaction.

A measurement and/or analysis system for the real-time quantitative PCRof the present invention, said real-time quantitative PCR including themethod of the present invention for the analysis of the melting curve ofa nucleic acid added to the above-described novel method of the presentinvention for the analysis of data obtained by a PCR method, also fallswithin the technical scope of the present invention.

Further, a seventh feature resides in a computer-readable recordingmedium with the individual procedures of the method of the presentinvention for the analysis of the melting curve of a nucleic acidrecorded therein as a program such that the procedures can be performedby a computer or a computer-readable recording medium with theindividual procedures of the method of the present invention for theanalysis of data obtained by a PCR method recorded therein as a programsuch that the procedures can be performed by a computer, wherein aprogram designed to make the computer perform the individual proceduresof the method of the present invention for the analysis of the meltingcurve of the nucleic acid is additionally recorded.

The above-described data analysis methods, systems and recording mediaof the present invention can be used in a variety of fields such asmedicine, forensic medicine, anthropology, paleontology, biology,genetic engineering, molecular biology, agricultural science andphytobreeding. They can be suitably applied to microorganism systemscalled “co-cultivation systems of microorganisms” or “symbioticcultivation systems of microorganisms”, in each of which various kindsof microorganisms are contained together or a microorganism and otheranimal- or plant-derived cells are contained together and cannot beisolated from each other. The term “microorganisms” as used herein meansmicroorganisms in general sense, and no particular limitation shall beimposed thereon. Illustrative are eukaryotic microorganisms, prokaryoticmicroorganisms, mycoplasmas, viruses and rickettsias.

The vial count of a particular cell strain in a co-cultivation system ofmicroorganisms or a symbiotic cultivation systems of microorganisms canbe determined by determining the number of copy(ies) of the 5S rRNA, 16SrRNA or 23S rRNA of the particular cell strain or its gene DNA in thesystem by using one or more of the above-described data analysismethods, systems and recording media of the present invention, becausethe number of copy(ies) of the gene DNA of 5S rRNA, 16S rRNA or 23S rRNAis specific to each cell strain. In the present invention, the vialcount of a particular cell strain can also be determined by applying thereal-time quantitative PCR of the present invention to a homogenate of aco-cultivation system of microorganisms or a symbiotic cultivationsystems of microorganisms. It shall also be noted that this method alsofalls with the technical scope of the present invention.

EXAMPLES

The present invention will be illustrated in further detail withreference to several examples and comparative examples below.

Example 1

Preparation of Nucleic Acid Probe:

Assuming that a target nucleic acid A comprises anoligodeoxyribonucleotide having a base sequence of (5′)tgc cat ccc ctcaat gg(3′), a nucleic acid probe according to the present invention wasprepared in the following manner.

Designing of Nucleic Acid Probe:

Based on the base sequence of the target nucleic acid, the nucleic acidprobe could be easily designed as an oligodeoxyribonucleotide having abase sequence of (5′)cca ttg agg gga tgg ca(3′). The nucleic acid probeof the present invention was designed in the following manner. Thenucleic acid probe was to be labeled with a donor dye BODIPY FL(Molecular Probes Inc., USA) at a phosphate group at the 5′ end and tobe labeled with an acceptor dye BODIPY 581/591 (Molecular Probes Inc.,D-2228, USA) at a OH group on the carbon atom at the 6-position of thebase ring of thymine, the fourth base from the 5′ end. The designednucleic acid probe 1 was BODIPY FL-(5′)cca (BODIPY 581/591)ttg agg ggatgg ca(3′).

Initially, the phosphate group of cytidylic acid was modified with alinker —(CH₂)₆—SH using 5′ Amino-Modifier C6 Kit (Glen Research Corp.,USA). The OH group on the carbon atom at the 6-position of the base ringof thymine was modified with a linker —(CH₂)₇—NH₂ using Amino-ModifierC2dT Kit (Glen Research Corp., USA). Using these modified cytidylic acidand thymidine, an oligonucleotide having the following base sequence wasprepared synthetically using a DNA synthesizer (ABI 394; PerkinElmerJapan Co., Ltd.). The prepared oligonucleotide was adeoxyribooligonucleotide having a base sequence: HS(CH₂)₆-(5′)cca(H₂N—(CH₂)₇-)ttg agg gga tgg ca(3′), in which the phosphate group at the5′ end was modified with the linker —(CH₂)₆—SH, and the OH group on thecarbon atom at the 6-position of the base ring of thymine, the fourthbase from the 5′ end, was modified with the linker —(CH₂)₇—NH₂. The DNAwas synthetically prepared according to a beta-cyanoethylphosphoramidite(2-cyanoethylphosphoramidite) method with 5′ end Tr ON. Afterpreparation, the protecting group was deprotected by a treatment with28% aqueous ammonia at 55° C. for 5 hours.

Purification of Prepared Product:

The above-prepared synthetic oligonucleotide-containing solution wasconcentrated to dryness. The dried residues were dissolved in 0.5 MNaHCO₃/Na₂CO₃ buffer (pH 9.0). The resultant solution was subjected to agel filtration method using an NAP-10 column (a product of PharmaciaCo.) to thereby remove unreacted materials.

Labeling with Acceptor Dye:

The filtrated product was dried to dryness and the dried residues werethen dissolved in 150 μL of sterile water (oligonucleotide A solution).A total of 1 mg of BODIPY 581/591-NHS (Molecular Probes Inc., USA) wasdissolved in 100 μL of dimethylformamide (DMF) and the solutioncontaining BODIPY 581/591 NHS was subjected to addition of theoligonucleotide A solution (150 μL) and 150 μL of 1 M NaHCO₃/Na₂CO₃buffer and mixing after the addition, followed by mixing by stirring atroom temperature vernight.

Purification of Prepared Product:

The resultant mixture was subjected to a gel filtration method by usingan NAP-25 (a product of Pharmacia Co.) to remove unreacted materials.Next, the protective group was deprotected with 2% TFA. The resultantwas subjected to a reversed phase HPLC method using a SEP-PAC C₁₈ columnto obtain a fraction containing a target product comprising the targetedoligonucleotide with the acceptor dye BODIPY 581/591 bound to the linker—(CH₂)₇—NH₂ of the oligonucleotide. The obtained fraction was subjectedto a gel filtration method using a NAP-10 (a product of Pharmacia Co.).

Labeling with Donor Dye:

The gel-filtrated product-containing fraction was concentrated todryness and the dried residue was dissolved in 150 μL of sterile water(oligonucleotide B solution). A total of 1 mg of BODIPY FL-Chloride(Molecular Probes Inc., USA) was dissolved in 100 μL of DMF and theresultant solution was then subjected to addition of the oligonucleotideB solution and 150 μL of 1 M NaHCO₃/Na₂CO₃ buffer, followed by overnightreaction at room temperature after stirring. Thereby the oligonucleotidewith the donor dye BODIPY FL bound to the linker —(CH₂)₆—SH at the 5′end was prepared.

Purification of Prepared Product:

The above reacted product was subjected to a gel filtration method usinga NAP-25 (Pharmacia Co.) to remove unreacted materials, and the obtainedfiltrated product was then subjected to a reversed phase HPLC method inthe same manner as above and thereby yielded nucleic acid probe 1labeled with a donor dye and acceptor dye according to the presentinvention. Nucleic acid probe 1 was an oligonucleotide labeled with theacceptor dye BODIPY 581/591 at the thymine base, the fourth base fromthe 5′ end, and with the donor dye BODIPY FL at the phosphate group atthe 5′ end. Incidentally, the nucleic acid probe of the presentinvention was eluted after the elution of the oligpnucleotide labeledwith the acceptor dye alone.

The amount of the nucleic acid probe of the present invention wasdetermined at 260 nm using a spectrophotometer. The wavelengths of thespectrophotometer was scanned at 650 to 220 nm for the absorptivity thatshowed absorption of BODIPY FL, BODIPY 581/591, and a DNA. The purity ofthe purified finnal product was assayed by reversed phase HPLC in thesame manner as above; the purified product showed a single peak.

The condition of the reversed phase chromatography was as follows:

-   Elution solvent A: 0.05 N TEAA 5% CH₃CN-   Elution solvent B: (for gradient): 0.05 N TEAA 40% CH₃CN-   Column: SEP-PAK C18; 6×250 mm-   Elution rate: 1.0 ml/min-   Temperature: 40° C.-   Detection: 254 nm

Example 2

Preparation of Probe Labeled with Acceptor Dye BODIPY 581/591 Alone((5′)cca (BODIPY 581/591)ttg agg gga tga ca(3′)) (Nucleic Acid Probe 2):

Nucleic acid probe 2 was prepared by the same method as that of nucleicacid probe 1, except for the procedures for binding the donor dye BODIPYFL to the phosphate group at the 5′ end.

Example 3

Preparation of Probe Labeled with Donor Dye BODIPY FL Alone ((BODIPYFL-(5′)cca ttg agg gga tgg ca(3′)) (Nucleic Acid Probe 3):

A nucleic acid probe was prepared by the same method as that of nucleicacid probe 1, except for the procedures for binding the acceptor dyeBODIPY 581/591 to the phosphate group at the 5′ end.

Example 4

Preparation of Target Nucleic Acid:

An oligonucleotide having a base sequence of (5′)tgc cat ccc ctc aatgg(3′) was prepared in the same manner as tha in the preparation of theaforementioned oligonucleotide and thereby yielded a target nucleic acidfor use in the present invention.

Example 5

Fluorescence Intensity Determination of Reaction System in which TargetNucleic Acid was Hybridized with Probe of the Present Invention:

In a quartz cell (10 mm×10 mm in size; capacity: 4 mL) was placed 500 μLof a buffer [120 mM NaCl (final concentration: 30 mM), 120 mM Tris-HCl(final concentration: 30 mM; pH=7.2)], followed by addition of 1460 μLof sterile distilled water and then stirring. To the mixture was added8.0 μL of a 10 μM solution of nucleic acid probe 1 of the presentinvention or a solution of nucleic acid probe 2 (final concentration ofthe nucleic acid probe: 40 nM), followed by stirring. The mixture washeld at 30° C. and fluorescence intensities thereof were examined at anexcitation wavelength of 490 nm (8 nm wide) and a measuring fluorescencewavelength of 580 nm (8 nm wide). The reaction system was thencontrolled at 60° C. and was subjected to addition of 32.0 μL of a 10 μMsolution of the target nucleic acid (final concentration of the targetnucleic acid: 160 nM) and then stirring. Further, the fluorescenceentinsities thereof were examined. The reaction system was kept at 30°C. to thereby allow the nucleic acid probe to hybridize with the targetnucleic acid, and the fluorescence intensities were examined. Theresults are shown in Tables 1 and 2.

In these procedures, the temperature of a cell (reaction temperature)was controlled using a thermostatic cell holder, a low-temperaturecirculation apparatus with a program system for crystallization PCC-7000(a product of Tokyo Rika Kikai Co., Ltd.). The fluorescence intensitywas measured using a fluorophotometer LS 50B (a product of PerkinElmerInc.). TABLE 1 Changes in Fluorescence Intensity at 580 nm Before andAfter Hybridization (HYB) (excited at 490 nm) Before After Decrease inDecrease HYB HYB Fluorescence Percentage Test System (60° C.) (30° C.)Intensity (%) Nucleic acid probe 24.82 4.10 20.72 83.5 1 and targetnucleic acid Nucleic acid probe 0.51 0.55 −0.04 −107.8 2 and targetnucleic acidHYB: HybridizationDecrease in Fluorescence Intensity (Differential Decrease inFluorescence Intensity Between Before Hybridization and AfterHybridization): [Fluorescence intensity before hybridization] -[Fluorescence intensity after hybridization]Decrease Percentage: [Decrease in fluorescence intensity]/[Fluorescenceintensity before hybridization]

TABLE 2 Changes in Fluorescence Intensity at 510 nm Before and AfterHybridization (HYB) (excited at 490 nm) Before After Decrease inDecrease HYB HYB Fluorescence Percentage Test System (60° C.) (30° C.)Intensity (%) Nucleic acid probe 10.5 7.8 2.7 25.71 1 and target nucleicacid Nucleic acid probe 55.56 9.6 45.96 82.72 3 and target nucleic acidHYB: HybridizationDecrease in Fluorescence Intensity Differential Decrease in FluorescenceIntensity Between Before Hybridization and After Hybridization):[Fluorescence intensity before hybridization] - [Fluorescence intensityafter hybridization]Decrease Percentage: [Decrease in fluorescence intensity]/[Fluorescenceintensity before hybridization]

Table 1 shows the fluorescence intensity of the acceptor dye BODIPY581/591, and Table 2 shows the fluorescence intensity of the donor dye.

Table 1 indicates the followings:

The fluorescence intensity of the acceptor dye before hybridization inthe test system containing nucleic acid probe 1 and the target nucleicacid is much higher than that in the test system containing nucleic acidprobe 2 and the target nucleic acid. This is capable of being explainedby the phenomena that FRET occurs between the donor dye BODIPY FL andthe acceptor dye BODIPY 581/591 to thereby allow the acceptor dye toemit fluorescence in nucleic acid probe 1. In contrast, FRET does notoccur and fluorescence is not emitted in nucleic acid probe 2, since ithas no donor dye in its molecule.

After hybridization, the fluorescence intensity significantly decreasesin the test system containing nucleic acid probe 1 and the targetnucleic acid. In contrast, the fluorescence intensity changes little inthe test system containing nucleic acid probe 2 and the target nucleicacid. This is capable of being explained by the phenomena that nucleicacid probe 1 hybridizes with the target nucleic acid and thehybridization causes the donor dye to approach the G-C hydrogen bondcomplex; the energy of the donor dye transfers to the G-C hydrogen bondcomplex.

Table 2 indicates the followings:

The fluorescence intensity of the donor dye before hybridization in thetest system containing nucleic acid probe 1 and the target nucleic acidis much higher than that in the test system containing nucleic acidprobe 2 and the target nucleic acid shown in Table 1. This is capable ofbeing explained by the phenomena that FRET occurs between the donor dyeBODIPY FL and the acceptor dye BODIPY 581/591, and the energy of thedonor dye transfers to the acceptor dye in nucleic acid probe 1. Incontrast, the nucleic acid probe 3 shown in Table 2 has no acceptor dyein its molecule, thereby does not induce FRET and emits fluorescence.

After hybridization, the fluorescence intensity in the test systemcontaining nucleic acid probe 1 and the target nucleic acid decreaseslittle. This is capable of being explained by the phenomena that thedonor dye approaches the G-C hydrogen bond complex upon hybridizationwith the target nucleic acid, and the energy transfers to the G-Chydrogen bond complex rather than to the acceptor dye. The fluorescenceintensity in the test system containing nucleic acid probe 3 and thetarget nucleic acid markedly decreases. This is capable of beingexplained by the phenomena that the donor dye approaches the G-Chydrogen bond complex upon hybridization with the target nucleic acid,and the energy which has been consumed to emit fluorescence nowtransfers to the G-C hydrogen bond complex.

Tables 1 and 2 show that the nucleic acid probe of the present inventionis designed so as to decrease the fluorescence intensities of both thedonor dye and acceptor dye upon hybridization with the target nucleicacid.

Example 6

An oligodeoxyribonucleotide having a base sequence of (5′)aacgatgccatggatttgg(3′) as a target nucleic acid B was prepared in the same manneras in Examples 1, 2, 3 and 4.

Nucleic acid probe 4 according to the present invention that canhybridize with the target nucleic acid B was prepared in the same manneras in the aforementioned examples by labeling an oligonucleotide withX-rhodamine as an acceptor dye and with BODIPY FL as a donor dye.Nucleic acid probe 4 had the following structure: BODIPYFL-(5′)ccaaat(X-Rhodamine)ccat ggcatca tt(3′)

Fluorescence Intensity Determination in Reaction System ContainingNucleic Acid Probe 1 of the Invention Hybridized with Target NucleicAcid A and Nucleic Acid Probe 4 of the Invention Hybridized with TargetNucleic Acid B:

In a quartz cell (10 mm×10 mm in size; capacity: 4 mL) was placed 500 μLof a buffer [120 mM NaCl (final concentration: 30 mM), 120 mM Tris-HCl(final concentration: 30 mM; pH=7.2)], followed by addition of 1460 μLof sterile distilled water and then stirring. To the mixture was added8.0 μL of a 10 μM solution of nucleic acid probes 1 and 4 of the presentinvention (final concentration of the nucleic acid probes: 40 nM), andfollowed by stirring. The mixture was held at 45° C. and thefluorescence intensities were determined at 580 nm (5 nm wide) on theprobe 1 and at 610 nm (5 nm wide) on the probe 4 both at an excitationwavelength of 490 nm (8 nm wide). Next, 32.0 μL each of solutions of thetarget nucleic acids A and B at individual concentrations indicated inTable 3 were added to the mixture and was stirred. The fluorescenceintensities of the resultant mixtures were determined, and the resultsare shown in Table 3.

The same thermostatic cell holder, fluorophotometer, and other apparatusand procedures as in the above examples were employed herein. TABLE 3Changes in Fluorescence Intensity at 580 nm and 610 nm Before and AfterHybridization (HYB) (both excited at 490 nm) Target Nucleic Acid BeforeAfter Decrease in Decrease Test Concentration HYB HYB FluorescencePercentage System (μM) (45° C.) (45° C.) Intensity (%) 580 nm 0 110 1082 1.8 (Target 20 107 67 40 37.4 Nucleic 40 101 30 71 70.2 Acid A) 60 9822 76 77.7 610 nm 0 80 85 5 0.6 (Target 20 75 50 25 33.3 Nucleic 40 8028 52 65.0 Acid B) 60 76 18 58 76.3HYB: HybridizationDecrease in Fluorescence Intensity (Differential Decrease inFluorescence Intensity Between Before Hybridization and AfterHybridization): [Fluorescence intensity before hybridization] −[Fluorescence intensity after hybridization]Decrease Percentage: [Decrease in fluorescence intensity]/[Fluorescenceintensity before hybridization]

Table 3 shows that the fluorescence intensities at 580 nm and 610 nmdecreases after hybridization to a degree depending on the concentrationof the target nucleic acid. When excited at 490 nm, changes in thefluorescence intensity at 580 nm belongs to those of the probe 1 of thepresent invention, and those at 610 nm belongs to those of the probe 4of the invention. These results show that, by using the nucleic aciddetermination method and the nucleic acid probes of the presentinvention, plural types of target nucleic acids can be easily determinedin parallel even if the plural target nucleic acids are contained in oneassay system.

Industrial Applicability

The nucleic acid probes of the present invention have the aboveconfiguration, show markedly decreased fluorescence intensities of boththe donor dye and acceptor dye after hybridization with the targetnucleic acids and thereby enable easy determination of the targetnucleic acids even in trace amounts precisely in a short time.

Donor dyes must transfer their energy to a G-C hydrogen bond complex andare limited in their types. However, acceptor dyes do not require such aproperty, and any acceptor dyes can be used herein as long as they arecapable of receiving transferred energy. Accordingly, different nucleicacid probes emitting fluorescence in different colors (having differentfluorescence wavelengths) can be prepared using one donor dye (i.e., atone excitation wavelength) according to the present invention. Thisconfiguration simplifies the optical system for excitation in a nucleicacid analyzer for determining multiple types of nucleic acids, since thedevice (analyzer) is usable only by having one excitation laser. Inother words, the invention can simplify the design and preparation ofsuch a nucleic acid analyzer.

In addition, in even a simple nucleic acid analyzer having an excitationlaser at one wavelength, multiple types of nucleic acid probes can beutilized; thereby multiple types of target nucleic acids can be inparallel determined using one analyzer.

1. A novel nucleic acid probe for determining one or more types oftarget nucleic acids, comprising a single-stranded nucleic acid beinglabeled with plural fluorescent dyes, the fluorescent dyes comprising atleast one pair of fluorescent dyes to induce fluorescence resonanceenergy transfer (FRET), the pair of fluorescent dyes comprising afluorescent dye (a donor dye) capable of serving as a donor dye and afluorescent dye (an acceptor dye) capable of serving as an acceptor dye,wherein the nucleic acid probe has a base sequence and is labeled withthe fluorescent dyes such that the fluorescence intensity of theacceptor dye decreases upon hybridization with a target nucleic acid. 2.The novel nucleic acid probe for determining one or more types of targetnucleic acids according to claim 1, wherein the fluorescence intensitiesof the donor dye and acceptor dye decrease upon hybridization with thetarget nucleic acid.
 3. The novel nucleic acid probe for determining oneor more types of target nucleic acids according to one of claims 1 and2, wherein the fluorescent dye capable of serving as a donor dye isselected from BODIPY FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamine,and 6-TAMRA.
 4. The novel nucleic acid probe for determining one or moretypes of target nucleic acids according to any one of claims 1 to 3,which comprises one pair of the donor dye and the acceptor dye.
 5. Thenovel nucleic acid probe for determining one or more types of targetnucleic acids according to any one of claims 1 to 4, wherein the nucleicacid probe is labeled with the donor dye in an end region and has a basesequence designed such that, when the probe hybridizes with the targetnucleic acid at the end region, the target nucleic acid has at least oneG (guanine) in its base sequence as a first to third base from itsterminal base hybridized with the probe.
 6. The novel nucleic acid probefor determining one or more types of target nucleic acids according toany one of claims 1 to 5, wherein the nucleic acid probe has a basesequence designed such that that plural base pairs of a probe-nucleicacid hybrid in a region labeled with the donor dye constitute at leastone pair of G (guanine) and C (cytosine) upon the hybridization with thetarget nucleic acid.
 7. The novel nucleic acid probe for determining oneor more types of target nucleic acids according to any one of claims 1to 6, wherein the nucleic acid probe is labeled with the donor dye in a5′ end region inclusive of the 5′ end.
 8. The novel nucleic acid probeaccording to any one of claims 1 to 7, wherein the nucleic acid probe islabeled with the donor dye in a 3′ end region inclusive of the 3′ end.9. The novel nucleic acid probe for determining one or more types oftarget nucleic acids according to claim 7, wherein the nucleic acidprobe has a G or C base at the 5′ end and is labeled with the donor dyeat the 5′ end.
 10. The novel nucleic acid probe according to claim 8,wherein the nucleic acid probe has a G or C base at the 3′ end and islabeled with the donor dye at the 3′ end.
 11. A novel method for nucleicacid determination, the method comprising the steps of adding one ormore types of the nucleic acid probes according to any one of claims 1to 10 into an assay system containing one or more types of targetnucleic acids, the nucleic acid probe(s) being capable of hybridizingwith the target nucleic acid(s), being in the number of type(s) equal toor larger than that of the target nucleic acid(s), and emittingfluorescence in different colors; allowing the nucleic acid probe(s) tohybridize to the target nucleic acid(s); and determining differentialdecrease(s) in fluorescence intensity between before and afterhybridization at wavelength(s) in the number of type(s) equal to orlarger than that of the types of the nucleic acid probe(s).
 12. A kitfor determining one or more types of target nucleic acids in an assaysystem, wherein said kit includes or accompanied by a nucleic acid probeaccording to any one of claims 1 to
 10. 13. A method for analyzing ordetermining polymorphism and/or mutation of one or more types of targetnucleic acids in an assay system, which comprises the steps of addingone or more types of the nucleic acid probes according to any one ofclaims 1 to 10 into an assay system containing one or more types oftarget nucleic acids, the nucleic acid probe(s) being capable ofhybridizing with the target nucleic acid(s), being in the number oftype(s) equal to or larger than that of the target nucleic acid(s), andemitting fluorescence in different colors; allowing the nucleic acidprobe(s) to hybridize to the target nucleic acid(s); and determiningdifferential decrease(s) in fluorescence intensity between before andafter hybridization at wavelength(s) in the number of type(s) equal toor larger than that of the types of the nucleic acid probe(s).
 14. A kitfor analyzing or determining polymorphism and/or mutation of one or moretypes of target nucleic acids in an assay system, wherein said kitincludes or accompanied by a nucleic acid probe according to any one ofclaims 1 to
 10. 15. A method according to claims 11 for determining oneor more types of target nucleic acids in an assay system, or accordingto claims 13 for determining polymorphism and/mutation of one or moretypes of target nucleic acids in an assay system, wherein said targetnucleic acids are one or more types of nucleic acids from cells of amicroorganism obtained by single colony isolation or from cells of ananimal.
 16. A method according to claims 11 for determining one or moretypes of target nucleic acids in an assay system, or according to claims13 for determining polymorphism and/mutation of one or more types oftarget nucleic acids in an assay system, wherein said target nucleicacids are one or more types of nucleic acids contained in cells or ahomogenate of cells, wherein cells are ones from a co-cultivation systemof microorganisms or symbiotic cultivation system of microorganisms.